Dengue virus and West Nile virus are mosquito-borne flaviviruses with considerable potential as agents for bioterrorism. These positive-strand RNA viruses are also emerging pathogens involved in natural disease outbreaks, making their control a significant public health goal worldwide (especially dengue virus). In addition to vaccine development and anti-viral therapeutics aimed at viral structural proteins, there is a need to identify steps in the intracellular replication cycles of these flaviviruses that can be targeted by small molecule inhibitors. One such step is the replication of viral RNA, a process mediated by the viral RNAdependent RNA polymerase (NSs), most likely in conjunction with other non-structural viral proteins and host cell proteins. Thus, the assembly of viral RNA replication complexes provides unique protein-protein interfaces that once identified, can be utilized as targets for anti-viral therapeutics. The experiments outlined in this proposal involve novel approaches to study dengue virus and West Nile virus RNA replication dynamics in vitro, using a recently-developed high-resolution single molecule bio-detection technology that is based on alternating laser excitation (ALEX) fluorescence spectroscopy. This innovative technology will subsequently be implemented in assays to screen for small molecule inhibitors of flavivirus RNA synthesis. The proposed project will (i) develop a 3-color Excitation/4-color Emission (3cEx/4cEm) ALEX instrument prototype for analysis of single molecule dynamics;(ii) develop reagents and experimental approaches for in uifro flavivirus RNA replication studies using cytoplasmic extracts from mammalian and insect cells;(iii) identify novel anti-viral agents as specific inhibitors of flavivirus RNA replication. Overall, the highresolution power to monitor molecular interactions and flavivirus RNA replication dynamics using the proposed analytical methods and in vitro assays will greatly aid SAR (structure-activity relationship) studies for lead optimization using structure-guided rational drug design.